Herpes simplex virus type-2 (HSV-2) infections are lifelong, and a leading cause of genital ulcer disease worldwide. The clinical outcome of genital HSV-2 reactivation varies widely from completely asymptomatic to infrequent and short-lasting recurrences to frequent and severe genital ulcerations: reasons for this variability are not known. Using biopsies of genital skin and mucosa from human volunteers, we have shown that HSV-2 reactivation results in a long-term persistence of local immune responses. CD8 T cells persist at the dermal-epidermal junction (DEJ) contiguous to the sensory nerve endings where virions are released. The unique anatomical distribution suggests that local CDS T cells might play a pivotal role in rapid containment of viral infection in the periphery, thus influencing the clinical and virologic course of HSV-2 disease in humans. In the last grant cycle, we developed a cell-type specific laser capture microdissection (LCM) method to isolate individual CD8 T cells in situ and measure their activity. By using combined approaches of CD8-specific LCM, whole genome transcriptional profiling and TCR repertoire deep sequencing, we can now elucidate associations between tissue resident memory CD8 T cells and genital herpes disease severity in humans. In this project, we will investigate the association between HSV-2 disease severity and the quantity, quality and diversity of tissue resident memory CD8 T cells at the site of previous HSV-2 recurrence in humans. Our specific aims are: 1) to define whether the anatomic distribution, density, decay kinetics and the antiviral signature genes of tissue resident memory CD8 T cells differ in participants with mild versus severe genital HSV-2 diseases; 2) to define whether the T cell receptor (TCR) repertoire dynamics and antigenic specificity of tissue resident memory CD8 T cells are associated with genital herpes disease severity. We will obtain sequential biopsy tissues from patient cohorts with distinct disease outcomes: mild disease, defined as recurrence rate < 2 episodes per year and severe disease as recurrence rate > 6 episodes per year. This project will define the characteristics of a successful peripheral immune response to HSV-2 that can be harnessed as a potential correlate of immunity during vaccine development.